Hz-1 virus is a member of the family Baculoviridae (Wilson, 1991) and is the first invertebrate virus in which differential viral gene expression during productive and persistent viral infections is demonstrated (Chao, et al., 1992). Thus far, two subfamilies have been described in the Baculoviridae insect virus family: the Eubaculovirinae and the Nudibaculovirinae.
The characteristic feature of Eubaculovirinae is the occlusion of virion in polyhedra proteinaceous inclusion body. The Eubaculovirinae is further divided into two genera: the first genus is "nuclear polyhedrosis virus", with Autographs californica multiple nuclear polyhedrosis (AcMNPV) as an exemplary type species of this genus; and, "granulosis virus" being the second genus.
Nudibaculovirinae, are known as nonoccluded viruses since virions are not packaged into inclusion bodies at any stage of their life cycle. (Wilson, 1991). Hz-1 virus was classified as a species of the Nudibaculovirinae family (Wilson, 1991). Recently Hz-1 virus and other non-occluded Baculoviruses were removed from the Baculovirus family and are temporarily unclassified (Volkman, 1995). Hz-1 virus was originally identified as a persistently infected virus in the Heliothis zea cell line, IMC-Hz-1 (Granados et al. 1978). Hz-1 virus is a rod-shaped virus containing 228 kb double-stranded circular DNA genome (Chao et al., 1990 a, b; Huang et al., 1982; Ralston et al., 1981). Relative to being a Baculovirus, the host range of Hz-1 virus is broad and persistent Hz-1 virus infections have been established in many insect cells (Chao et al., 1990a; Wood and Burand, 1986; Granados et al., 1978). Infection of Hz-1 virus can produce both productive and persistent viral infections (Ralston, et al., 1981; Burand et al., 1986; Chao et al., 1992).
More specifically, persistent viral infection has long been reported to occur naturally in insects. Many of the persistently infected viruses can be activated (Burand et al., 1986; Chao et al., 1985; Jurkovicoba, 1979; Hughes et al., 1993; Podgwaite and Mazzone, 1986; Wood and Burand, 1986) upon the change of rearing temperature, high humidity, decrease of food quality, superinfection of different viruses, and/or other stimuli. However, due to the difficulty in establishing persistent viral infection in laboratory insect stocks, persistent viral infection is usually studied after an unexpected viral activation from a previously healthy-looking insect or cell. It would therefore be advantageous to have a vector system in which persistent viral infection may be achieved, with activation or replication available if desired for virus propagation (e.g., cloning) or if necessary or desired for expression, such as of exogenous DNA.
The persistently infected Hz-1 virus was shown to be activated by heterologous viruses (Burand et al., 1986) and the host cells can be resistant to the superinfection of homologous virus due to induction of apoptosis (Lee et al., 1993).
Differential viral gene expression is also known in the herpes viruses in mammals. In the Epstein-Barr virus, approximately 12 genes are expressed during latent infection and approximately 50 to 100 viral genes were expressed in cells that have entered a lytic phase of viral growth (Klein, 1989; Metzenberg, 1990). In herpes simplex virus, the virus probably encodes more than 70 polypeptides during productive viral infection. During latent viral infection, only three related latent associated transcripts (LATS) generated by the same gene are detectable (Spivack and Fraser, 1987; Stevens et al., 1987).
The patent and scientific literature includes various viral vector systems, uses therefor, and exogenous DNA for expression of protein by such systems, as well as uses for such proteins and uses for products from such proteins.
For instance, recombinant poxviruses (e.g., vaccinia, avipox virus) and exogenous DNA for expression in viral vector systems can be found in U.S. Pat. No. 5,174,993 (e.g., recombinant avipox virus, vaccinia virus; rabies glycoprotein (G), gene, turkey influenza hemagglutinin gene, gp51, 30 envelope gene of bovine leukemia virus, Newcastle Disease Virus (NDV) antigen, FeLV envelope gene, RAV-1 env gene, NP (nudeoprotein) gene of Chicken/Pennsylvania/1/83 influenza virus, matrix and preplomer gene of infectious bronchitis virus; HSV gD; entomopox promoter, inter alia), U.S. Pat. No. 5,338,683, e.g., recombinant vaccinia virus, avipox virus; DNA encoding HSV glycoproteins, inter alia; U.S. Pat. No. 5,494,807 (e.g., recombinant vaccinia, avipox; exogenous DNA encoding antigens from rabies, Hepatitis B, JEV, YF, Dengue, measles, pseudorabies, Epstein-Barr, HSV, HIV, SIV, EHV, BHV, BVDV, HCMV, canine parvovirus, equine influenza, FeLV, FHV, Hantaan, C. tetani, avian influenza, mumps, NDV, inter alia); WO 92 08789 (e.g., recombinant vaccinia, avipox, Morbillivirus e.g., measles F, hemagglutinin, inter alia); U.S. Pat. No. 4,722,848 (e.g., recombinant vaccinia virus; HSV tk, glycoproteins e.g., Gb, Gd!, influenza HA, Hepatitis B e.g., HBsAg!, inter alia); U.K. Patent GB 2 269 820 B (recombinant poxvirus; flavivirus structural proteins); WO 92/22641 (e.g., recombinant poxvirus; immunodeficiency virus, inter alia); WO 93/03145 (e.g., recombinant poxvirus; IBDV, inter alia); WO 94/16716 (e.g., recombinant poxvirus; cytokine and/or tumor associated antigens, inter alia).
U.S. Pat. No. 4,769,331 relates to herpesvirus as a vector. There are also poliovirus and adenovirus vector systems.
Baculovirus expression systems, exogenous DNA for expression therein, and purification of recombinant proteins therefrom can be found in Richardson, C. D. (Editor), Methods in Molecular Biology 39, "Baculovirus expression protocols" (1995 Humana Press Inc.) (see, e.g., Ch.18 for influenza HA expression, Ch.19 for recombinant protein purification techniques), Smith et al., "production of huma beta interferon in insect cells infected with a Baculovirus expression vector," Molecular and Cellular Biology, December, 1983, Vol. 3, No. 12, p. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus Vector," Molecular and Cellular Biology March 1984, Vol. 4, No. 3, pp. 399-406; EPA 0 370 573 (Skin test and test kit for AIDS, discussing Baculovirus expression systems containing portion of HIV-1 env gene, and citing U.S. application Ser. No. 920,197, filed Oct. 16, 1986 now abandoned and EP Patent publication No. 0 265785); U.S. Pat. No. 5,147,788 (Baculovirus vectors and methods of use); U.S. Pat. Nos. 5,155,037, 5,278,050 (insect signal sequences replacing CD4 natural signal peptide coding in recombinant Baculovirus expression vector); U.S. Pat. No. 5,162,222 (Baculovirus first IE promoter/Baculovirus second IE promoter (opposite orientation to first)/cloning restriction site (for insertion of heterologous coding DNA sequence Baculovirus vector); U.S. Pat. No. 5,244,805 (Baculovirus expression vectors); U.S. Pat. No. 5,169,784 (Baculovirus dual promoter expression vector); U.S. Pat. No. 5,194,376 (Baculovirus expression system); U.S. Pat. No. 5,322,774 (procaryotic leader sequence in recombinant Baculovirus expression system); U.S. Pat. No. 5,110,729 (peptide production using Baculovirus in cultured cells); U.S. Pat. No. 5,179,023 (Baculovirus expression vectors expressing recombinant human alpha-Galactosidase in cell cultures); U.S. Pat. No. 5,186,933 (method to express rotavirus gene in Baculovirus); U.S. Pat. No. 5,229,293 (Baculovirus-JEV recombinant; JEV E protein as exogenous DNA); U.S. Pat. No. 5,260,199 (1,25-dihydroxyvitamin D.sub.3 receptor protein produced in Baculovirus); U.S. Pat. No. 5,290,686 (influenza A M2 expression in Baculovirus); U.S. Pat. No. 5,294,548 (hepatitis A virus expression in Baculovirus); U.S. Pat. No. 5,272,063 (human nerve growth factor expression in Baculovirus); U.S. Pat. Nos. 5,180,581 and 5,352,451 (Baculovirus with inactivated gene optionally as modified to express A protein as insect control agent); U.S. Pat. No. 5,300,435 (cell line susceptible to Baculovirus); and U.S. Pat. No. 5,179,007 (production and isolation of protein through lepidopteran cells transfected or infected with recombinant Baculovirus).
Baculovirus vector systems offer unique advantages. For instance, the advantages of: eliminating non-specific reactions from human cell cultures, having a relatively narrow host range which is restricted to arthropods, and, of having U.S. EPA approval of use of species thereof for control of insect pests (with Autoggrapha californica nuclear polyhedrosis virus ("AMNPV") having been applied to crops for many years under EPA Experimental Use Permits).
However, Baculovirus vector systems have suffered from disadvantages such as reliance upon the very late polyhedrin promoter. That is, the polyhedrin promoter based Baculovirus expression system ("PPBE") or, the use of the polyhedrin promoter, has disadvantages. For instance, the polyhedrin promoter is so very late as a promoter, expression can occur 15 to 18, average 16, hours after infection when nucleocaspids are enveloped within the nucleus and viral occlusions begin to form (each occlusion containing many viral particles embedded in a paracrystalline protein matrix, formed from polyhedrin, a single matrix, which accumulates to high levels and can constitute 25% or more of total protein mass in infected cell).
Further, Baculovirus is a lytic virus. That is, lyses of the host cells occurs. This lyses of host cells can complicate production and purification of proteins not naturally occurring in Baculovirus. Lysis results in the release of numerous host and viral proteins, as well as loss of control of the enzymatic system of the host cells. This release and loss of control can lead to degradation on any structural level of foreign protein expressed with Baculovirus. Accordingly, harvesting of foreign proteins must be carefully timed.
Thus, the possibility of avoiding lysis would be advantageous. It would therefore be advantageous to have a strong and early to very early promoter. It would even be more advantageous to have an early to very early promoter which is stronger or has greater expression than polyhedrin. It would also be advantageous to provide such a strong to very strong, early to very early promoter which can be used in the polyhedrin coding region of typical Baculovirus presently used, e.g., AcMNPV, since the polyhedrin coding region is an already known non-essential site in Baculovirus, or even in other viral vector systems. It would even be further advantageous if rather than lysis there was the possibility even of persistent infection such that through cell life and replication there is expression of the foreign protein. It would additionally be advantageous to provide nucleic acid molecules encoding such a strong to very strong, early to very early promoter, or functional portions thereof, or, nucleic acid molecules which hybridize to the promoter or functional portion thereof, so as to be useful for detecting presence or for PCR amplification.
However, given the similarity in replication between herpes virus and Baculovirus (nuclear replication) and of a latency or persistent infection, use of pag1, the promoter thereof, and fragments thereof, especially operably linked to exogenous DNA, in herpes virus, are envisioned.